SUPPLEMENTARY INFORMATION
PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein
Kinase C signaling
Martin Mueller1,2, Andreina Schoeberlein3, Jichun Zhou1,4, Marianne Joerger-Messerli3,
Byron, Oppliger3, Ursula Reinhart3, Angelique Bordey5, Daniel Surbek2,3, Eytan R Barnea6,7,
Yingqun Huang1, Michael Paidas1,8
SUPPLEMENTARY METHODS
Antibodies, siRNAs, inhibitors, and peptides
The antibodies against TLR4 (Abcam, ab22048), active CASP-3 (Cell Signaling, 9661S),
BAD (Cell Signaling, 9239), p-BADSer112 (Cell Signaling, 5284P), GAP-43 (Abcam,
ab11136), p-GAP-43Ser41 (Santa Cruz, sc-17109), CREB (Cell Signaling, 9197), pCREBSer133 (Cell Signaling, 9198), phospho-PKC Substrate (Cell Signaling, 6967),
phospho-PKA Substrate (Cell Signaling, 9624), β-Actin (Cell Signaling, 4970), and β-Tubulin
(Abcam, ab6046) were purchased. The siRNAs for TLR4 (Thermo Scientific Dharmacon, L008088-01) and the negative control siRNA (Thermo Scientific Dharmacon, D-001810-1020) were purchased. LPS (Sigma Aldrich, L6529-1MG), inhibitor H89 (EMD Millipore,
371963-1MG), and inhibitor Gö6983 (EMD Millipore, 365251) were purchased. sPIF
(MVRIKPGSANKPSDD)
and
PIFscr
(GRVDPSNKSMPKDIA)
with
>95%
purity
documented by HPLC and mass spectrometry, were generated at Biosynthesis (Lewisville,
Tx, USA).
TLR4 siRNA knockdown
Cells were transfected in a 48-well plate scale. To prepare siRNA transfection solution for
each well, 16pmol of siCon or siRNA was mixed with 50 μl OPTI- MEM (Gibco 31985-070)
by gentle pipetting. In parallel, 0.5 μl Lipofectamine 2000 was mixed with 50 μl OPTI-MEM.
Following 5 min of incubation at room temperature (RT), the two solutions were mixed by
gentle pipetting and incubated for 30 min at RT to allow the formation of siRNA/lipid
complexes. At the end of incubation, the 100 μl transfection solution was used to re-suspend
cell pellet (8×104 cells). After incubation at RT for 10 min, regular growth media was added
at a ratio of 1:4 (1 volume of transfection solution / 4 volumes of growth media) and the cell
suspension was transferred to the culture plate. After 24 h incubation at 37 °C in 5% CO 2, the
medium was replaced with serum free media containing LPS (0.5μg/ml) for 24 hours. RNAs
and proteins were extracted and analyzed at the indicated time points following transfection
and/or peptide treatment.
Western Blot analyses
These were used to assess TLR4, active CASP-3, BAD, p-BADSer112, GAP-43, p-GAP43Ser41, CREB, and p-CREBSer133 protein levels in cultured cells and rat brain tissues.
Briefly, cell pellets or frozen tissue samples were quickly lysed in ~10 volumes of 2x SDSsample buffer heated at 100 °C for 5 min. To homogenize cell pellets, occasional vortexing
was performed. To homogenize brain tissues, lysate was passed through 20G needles 10 times
for each sample. Five to 10 μl homogenized samples were separated on a 10% SDS
polyacrylamide gel, followed by Western blot analyses. Whole brain lysate for kinome
profiling (Phospho-PKA/PKC Substrates) was isolated following a protocol developed by
Cell Signaling Technology. Briefly, tissue was homogenized in lysis buffer (20mM HEPES
pH 8.0, 9M urea, 1mM sodium orthovanadate, 2.5mM sodium pyrophosphate, 1mM βglycerophosphate), sonicated, and cleared by centrifugation. Protein concentration was
measured using the Bradford assay and a total of 30 μg protein was loaded for each lane.
Protein bands on Western gels were quantified using Image J (US National Institutes of
Health).
qRT-PCR
These were carried out as previously described (1). For mRNA quantification, total RNAs
were extracted from cells using PureLink RNA Mini Kit (Ambion, catalog number
12183018A). cDNA was synthesized using Bio-Rad iSCRIPT kit (1725122) in a 20 μl
reaction containing 500-800 ng of total RNA. Real-time PCR was performed in a 15 μl
reaction containing 1ul of cDNA using iQSYBRGreen (Bio-Rad) in a Bio-Rad iCycler. PCR
was performed by initial denaturation at 95°C for 5 min, followed by 40 cycles of 30 sec at
95°C, 30 sec at 60°C, and 30 sec at 72°C. Specificity was verified by melting curve analysis
and agarose gel electrophoresis. The threshold cycle (Ct) values of each sample were used in
the post-PCR data analysis. The indicated mRNA levels were normalized against Tubulin.
The PCR primers for the mouse and rat mRNAs are listed below.
Mouse:
Beta-tubulin:
Forward
5′-CGTGTTCGGCCAGAGTGGTGC,
GGGTGAGGGCATGACGCTGAA;
TLR4:
Forward
Reverse
5′-
AGACCTCAGCTTCAATGGTG,
Reverse GAGACTGGTCAAGCCAAGAA; Bad: Forward 5'- AGT CTT TCG AGG CCT
TAG GA -3', Reverse 5'- CCC CAG TTA TGA CAG GAC AG -3'; Bcl2: Forward 5'- CTG
GAA ACC CTC CTG ATT TT -3', Reverse 5'- AAA TAT TTC AAA CGC GTC CA -3';
Gap43: Forward 5'- AGG GAG ATG GCT CTG CTA CT -3', Reverse 5'- GAG GAC GGG
GAG TTA TCA GT -3': Casp3: Forward 5'- GTC CAT GCT CAC GAA AGA AC -3',
Reverse 5'- ACC TGA TGT CGA AGT TGA GG -3'; Bdnf: Forward 5'- GGT GCA GAA
AAG CAA CAA GT -3', Reverse 5'- GCA CAA AAA GTT CCC AGA GA -3'.
Rat:
Beta-tubulin
Forward:
5′-CGTGTTCGGCCAGAGTGGTGC,
Reverse:
5′-
GGGTGAGGGCATGACGCTGAA Bcl2: 5'- CAT CAC TCT GGG TGC ATA CC -3',
Reverse 5'- TTG ACC ATT TGC CTG AAT GT -3', Bdnf: Forward 5'- CAT TTC ATG ACA
CTC GTG GA -3', Reverse 5'- ATT TCA GTG GCA GTG TGG AT -3'; Gap43: Forward 5'CAG GAA AGA TCC CAA GTC CA -3', Reverse 5'- GAA CGG AAC ATT GCA CAC AC
-3'; Bad: Forward Primer 5'- CCT TGC TTT GGA GTT TTC AA -3', Reverse Primer 5'AAA GCA CGT TTC TTG ACC TG -3'.
Perioperative Care of the animals:
Perioperative care was performed in accordance with Ethics Committee and Veterinary
Department of the Canton of Berne, Switzerland guidelines. Acclimatization of animals to the
laboratory environment was allowed prior to surgery. Aseptic rodent survival surgery
guidelines were followed. We injected Buprenorphine (0.01-0.1 mg/kg body weight)
subcutaneously 30 min prior incision as a pre-operative procedure once prior to surgery.
Buprenorphine was used as postoperative analgesia (starting 4-6 hours post-surgery, every 812 hours for 2-3 days) as well. We used Isoflurane as anesthetic (Induction with 4% and
continuation with 1.5-2%; 3l/min). Adequate depth of anesthesia was confirmed. After
surgery, animals were placed back to the mother as soon as they were awake, able to right
themselves and able to move about. We monitored several parameters including anesthetic
depth (persistence), clinical signs and condition. We monitored the pups every hours for at
least 6-8 hours post operation with special attention to breathing pattern and mobility, as well
as signs of seepage or bleeding from the incision. Once completely recovered, the dam was
transferred back to the room where the rats are housed. When neonates have reached the age
of interest animals were sacrificed by Natrium-Pentothal (100mg/kg body weight i.p.), cardiac
perfusion, and decapitation.
Immunohistochemistry:
We performed cardiac perfusion with PBS followed by formaldehyde (4%; Merck,
Whitehouse Station, NJ) before brain harvesting. Brains were removed surgically and fixed in
formaldehyde solution (4%) for 2-4 hours at room temperature (RT) followed by 4°C for a
total time of 24-48 hours. Fixed brains were embedded in paraffin and sectioned into 7µm
slices. After deparaffinization of the slides, the target was retrieved in citrate buffer (10 mM;
pH 6.0) in a pressure cooker for 15 minutes. Slides were washed in 0.1% Tween-20/PBS and
blocked in 10% goat serum/1% bovine albumin/PBS. Neurons were detected by a mouse
monoclonal antibody specific for the neuronal nuclear antigen (NeuN, Chemicon/Millipore,
MAB377, 1:100). Anti-active + pro Caspase-3 was detected by a mouse monoclonal antibody
(CASP-3, Abcam, ab13847, 1:100). A rabbit polyclonal antibody against the homeobox
protein cut-like 1 (CUX1, Santa Cruz Biotechnology, sc-13024, 1:100), a highly specific
marker for superficial layer neurons, was used to visualize neuronal migration. Following first
antibody incubation, slides were washed in 0.1% Tween-20/PBS (2x 5min) and incubated in
endogenous peroxidase blocking solution at RT for 15 min. Peroxidase-labeled polymer
(DAKO anti mouse or anti rabbit) was applied to the slides for 30 min at RT. Slides were
washed in PBS (3x5 min), followed by application of DAB+ chromogen in buffer substrate
for 10-30 min, according to the manufacturer’s instructions (DAKO EnVision+ System-HRP
(DAB), K4007). Slides were rinsed in ddH2O, counterstained in Cresyl violet (Nissl body
staining for neuronal structure and gross brain morphology) dehydrated in a series of ethanol
baths (95% >100%) and xylene, and mounted with Eukitt (Sigma-Aldrich, St. Louis, MO).
REFERENCES
1.
Mueller M, et al. (2014) PreImplantation factor promotes neuroprotection by targeting
microRNA let-7. Proceedings of the National Academy of Sciences of the United
States of America 111(38):13882-13887.